The mosquito-borne flavivirus family includes human pathogens such as yellow fever virus (YFV), West Nile virus (WNV), and the four serotypes of Dengue (DEN) viruses. These viruses, included in the NIAID list of Class A, B, or C human pathogens, cause serious illnesses associated with considerable morbidity and mortality and these diseases have emerged in recent years. Since the first cases of WNV infections in the Western hemisphere were recorded in New York city in 1999, the WNV has become a rapidly spreading major public health concern throughout the U.S. One of the long tern goals of this laboratory has been to develop antiviral therapeutics through understanding of key pathways in the viral lifecycle through development of in vitro systems that mimic processes these viruses use in their hosts. For example, polyprotein processing has been identified as a key early event that is crucial for the viral life cycle. The two-component dengue viral serine protease, required in this process has been expressed in E. coli, purified, and biochemically and kinetically characterized. The crystal structures of the protease domain, alone or in complex with a serine protease inhibitor have been solved. The overall objective of this proposal is to purify the E. coli-expressed West Nile viral protease and characterize its biochemical and kinetic properties in the presence and absence of protease inhibitors. This objective will be achieved with the following Specific Aims. Aim 1: Purification and characterization of the two-component WNV NS3-protease.The protease will be purified from E. coli and determine the kinetic and biochemical parameters of the enzyme. The WNV protease has been expressed with an N-terminal or C-terminal His tag. The enzyme will be purified to near-homogeneity in two steps using metal affinity and gel filtration chromatography steps. The enzyme activity will be assayed using radiolabeled natural polypeptide precursor having the protease sensitive site, fluorogenic peptide substrates, or internally quenched (IQ) fluorogenic substrates and the kinetic parameters will be determined. Aim 2: Synthesis of novel novel heterocyclic scaffold core structure, covalent and non-covalent inhibitors of DEN and WNV proteases will be synthesized. This strategy has been successfully employed in development of chymotrypsin-and trypsin-like serine proteases and preliminary screen of DEN protease activity shows promising results. Aim 3. Analysis of potencies of serine protease inhibitors in vitro and in vivo. The in vitro assays with fluorogenic (and IQ) substrates will be carried out in the presence and absence of inhibitors. Product-based inhibitors, inhibitors similar to those developed for HCV protease, covalent and noncovalent inhibitors synthesized using the heterocyclic scaffold will be assayed. A cell-based assay will be developed for evaluating the inhibitory potencies of these compounds. Since there is no effective vaccine available for either DEN or WNV, this antiviral strategy is likely to yield lead inhibitors useful as therapeutic agents in the control of these lethal pathogens.